Inhibition of non-homologous end joining in Fanconi Anemia cells results in rescue of survival after interstrand crosslinks but sensitization to replication associated double-strand breaks

Abstract

When Fanconi Anemia (FA) proteins were depleted in human U2OS cells with integrated DNA repair reporters, we observed decreases in homologous recombination (HR), decreases in mutagenic non-homologous end joining (m-NHEJ) and increases in canonical NHEJ, which was independently confirmed by measuring V(D)J recombination. Furthermore, depletion of FA proteins resulted in reduced HR protein foci and increased NHEJ protein recruitment to replication-associated DSBs, consistent with our observation that the use of canonical NHEJ increases after depletion of FA proteins in cycling cells. FA-depleted cells and FA-mutant cells were exquisitely sensitive to a DNA-PKcs inhibitor (DNA-PKi) after sustaining replication-associated double strand breaks (DSBs). By contrast, after DNA interstrand crosslinks, DNA-PKi resulted in increased survival in FA-deficient cells, implying that NHEJ is contributing to lethality after crosslink repair. Our results suggest FA proteins inhibit NHEJ, since repair intermediates from crosslinks are rendered lethal by NHEJ. The implication is that bone marrow failure in FA could be triggered by naturally occurring DNA crosslinks, and DNA-PK inhibitors would be protective. Since some sporadic cancers have been shown to have deficiencies in the FA-pathway, these tumors should be vulnerable to NHEJ inhibitors with replication stress, but not with crosslinking agents, which could be tested in future clinical trials.

Keywords: Double strand break repair; Fanconi anemia; Homologous recombination; Interstrand crosslinks; Non-homologous end joining.

Figure 1

Depletion of FA proteins decreases both HR and m-NHEJ repair of an I-SceI induced DSB. (A) representative flow cytometry profiles of GFP (Q3; HR) vs RFP (Q1; m-NHEJ) expression in U2OS cells transfected with siRNA targeting various FA proteins (FANC-A, C, D2, G, or L), or NT control, following repair of an I-SceI induced DSB. (B) quantification of effect of FA protein depletion on HR, normalized to NT. (C), quantification of effect of FA protein depletion on m-NHEJ, normalized to NT. Error bars represent S.E.M. of at least 5 independent experiments. ** P<0.01 determined by student’s t-test.

Figure 2

Depletion of FA proteins leads to increased Ku-dependent NHEJ. (A) Schematic illustrating the experimental procedure. (B) Western blot confirming protein knock-down 48 h after siRNA treatment, and expression of complemented RAG1/RAG2. (C) RAG-induced NHEJ as measured by development of chloramphenicol resistance, normalized to NT. Error bars represent S.E.M. of at least 3 independent experiments. *P<0.05 as determined by student’s t-test.

Figure 3

FA-depleted U2OS cells are uniquely sensitive to replication-associated DSB causing agents after DNA-PKcs inhibition. (A and D) FA depletion (FANCD2 or FANCG) does not render cells significantly sensitive to etoposide (ETO; A), or camptothecin (CPT; D). (B and E) FA depletion (FANCD2 or FANCG) makes cells uniquely sensitive to ETO (B) or CPT (E) after DNA-PKcs inhibition (PKi; NU7441, 1 μM). (C and F) Sensitization of FA-depleted cells to ETO (C) or CPT (F) following DNA-PKcs inhibition, relative to NT. Error bars represent S.E.M of at least 3 independent experiments. n.s. not significant, *P<0.005 determined by student’s t-test.

Figure 4

FA-depleted U2OS cells are uniquely sensitive to IR after PARP inhibition. (A) FA depletion (FANCD2 or FANCG) has no significant effect on cell survival following irradiation. (B) FA depletion (FANCD2 or FANCG) confers sensitivity to irradiation after PARP inhibition (veliparib, 40uM). (C) sensitization of FA-depleted cells to irradiation following PARP inhibition, relative to NT. Error bars represent S.E.M of at least 3 independent experiments. n.s. not significant, *P<0.05 determined by student’s t-test.

Figure 5

FA depleted/deficient cell sensitivity to MMC is rescued by inhibition of DNA-PKcs. (A) FA-depleted (FANCD2 or FANCG) U2OS cells are sensitive to MMC exposure. Left; non-target siRNA-treated cells do not show additional sensitivity or rescue from MMC exposure after DNA-PKcs inhibition (PKi; NU7441 1 μM), with FANCD2/G shown for comparison. Middle and right; FANCD2- and FANCG-depleted cells, respectively, demonstrate a partial rescue of sensitivity to MMC following DNA-PKcs inhibition (PKi; NU7441, 1μM). (B) Rescue of FA-depleted cells by DNA-PKcs inhibition after MMC exposure, relative to DMSO control. (C) Cell viability assay demonstrating FANCG-deficient cells rescue of sensitivity of MMC exposure by DNA-PKcs inhibition, in comparison to the wild-type complemented cell line.

Figure 6

FA proteins regulate recruitment of DSB repair proteins to sites of replication-associated DNA damage in U2OS cells. (A) Representative immunofluorescence images of BRCA1 (green), Mre11 (green), phosphorylated DNA-PKcs (pDNA-PKcs; red), and 53BP1 (red) foci in response to aphidicolin treatment (25 μg/ml, 4 hours) after depletion of FANCD2 or FANCG. (B–E) quantification of immunofluorescence images demonstrating protein response of BRCA1, MRE11, pDNA-PKcs, and 53BP1 to indicated treatments. Error bars represent S.E.M. of at least 3 independent experiments, at least 250 cells counted per experiment. n.s. not significant, * P<0.05, ** P<0.01, determined by student’s t-test.